The overall hypothesis of this proposal is that anandamide (an endogenous ligand that binds to cannabinoid and vanilloid receptors) is taken up into cells by a mechanism of simple diffusion rather than, as currently believed, by an anandamide transporter. Examining approximately a dozen immortalized and primary cell lines reported in the literature to have an anandamide transporter, the specific aims of the proposal are: 1) to determine if anandamide uptake occurs by simple diffusion (non-saturable) or facilitated diffusion (saturable). These experiments will differ from other published reports in that uptake of anandamide will be studied at initial times (25 seconds). These initial rates will distinguish actual uptake from downstream events such as metabolism or intracellular sequestration, which may give the appearance of saturation and an anandamide carrier. 2) to investigate the role of the "anandamide transport inhibitors" described in the literature. These "anandamide transport inhibitors" will be tested as uptake competitors using initial uptake rates as opposed to the prevailing studies in the literature that employed times greater than one minute that measures net uptake. If our hypothesis is correct, none of these will inhibit transport but will, in fact, be shown to inhibitor FAAH. Therefore the approach being used by other investigators in the field to synthesize "anandamide transport inhibitors" may be flawed from a theoretical viewpoint. 3) to elucidate the cellular distribution of FAAH with particular emphasis on its intracellular localization. If immunofluorescence indicates that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, it suggests that anandamide hydrolysis will appear as a downstream event that will be confirmed by the observed kinetics of anandamide hydrolysis in Specific Aims 1 and 2. In contrast to prevailing studies, this proposal will unequivocally determine if anandamide uptake is a process of simple diffusion. The clarification of the mechanism of anandamide inactivation is important for the development of therapeutics in areas such as drug addiction, mood, pain, fertility, and appetite regulation. [unreadable] [unreadable]